We studied the role of sodium transport and calcium channels in protein kinase C mediated signal-transduction pathways in bovine airway smooth muscle. 4-beta phorbol 12,13 dibutyrate (PDB), an activator of protein kinase C, caused dose-related slowly developing contraction in bovine bronchial rings with a peak effect at 60-90 min. 4-alpha PDB, an inactive analogue, was without effect. Mean peak PDB-induced contraction (measured as a percentage of the maximum response to methacholine) was reduced from 122% to 20% when experiments were carried out in calcium-free fluids +EDTA (10(-3) M). Similar reductions were seen in the presence of nifedipine and verapamil, inhibitors of voltage-dependent calcium channels. Amiloride at high concentrations (10(-3) M) reduced the contractile response to PDB from 87% to 20%, but at a concentration which inhibits the sodium entry channel (10(-6) M), amiloride was without effect. 5-N,N-hexamethylene amiloride (10(-5) M), a specific inhibitor of Na+/H+ exchange, did not alter the contraction produced by PDB. Frusemide (10(-5) M), an inhibitor of Na(+)-K(+)-Cl- cotransport, was without effect on PDB contractions. We conclude that phorbol ester-induced contraction of bovine airway smooth muscle is dependent on calcium entry via voltage-dependent calcium channels but is independent of Na+ entry, Na+/H+ exchange or Na(+)-K(+)-Cl- cotransport.